The present invention relates to the use of lyophilized active tubulin. More particularly, the present invention relates to the diagnostic and therapeutic applications of lyophilized active tubulin, and the use of lyophilized active tubulins in the fields of drug discovery and research.
Tubulin is an essential intracellular protein that is necessary for mitosis, transport of intracellular material, cell structure, and cell motility. Tubulin is composed of a heterodimer of two closely related 55 Kilodalton proteins called alpha and beta tubulin. These two proteins are encoded by separate genes or small gene families, whose sequences are highly conserved throughout the eukaryotic kingdom.
Tubulin polymerizes to form structures called microtubules. When tubulin polymerizes it initially forms protofilaments. Microtubules consist of 13 protofilaments and are 25 nm in diameter, each xcexcm of microtubule length being composed of 1650 tubulin heterodimers. Microtubules are highly ordered fibers that have an intrinsic polarity. There is a dynamic flux between microtubules and tubulin. When this equilibrium is perturbed by anti-tubulin agents like paclitaxel (taxol), cells will arrest in mitosis and eventually die. (Schiff et al., 1980, Proceedings of the National Academy of Sciences USA, 77, 1561-1565.) This is the mechanistic basis of how anti-tumor (TAXOL, vinblastine, and nocodazole ), anti-fungal (benomyl) and anti-anthleminitic (mebendazole) agents interact with tubulin. Vinblastine, paclitaxel (TAXOL(trademark) Bristol Myers-Squibb Co.), and nocodazole bind to tubulin and thus inhibit mitosis (Weisenberg, R. C., and S. N. Timasheff, 1970, Biochemistry, 21, 4110-4116; Schiff et al., 1980). These agents were identified through cell or organism based screens and were only later found to interact with tubulin.
Different forms of tubulin have been isolated. These include a microtubule associated protein (MAP)-rich tubulin that is 50% to 97% purified (Shelanski, M. L., Gaskin, F., and C. R. Cantor, 1973, Proceedings of the National Academy of Sciences USA, 70, 765-768), highly purified (97% to 99.99% or apparently 100% purified by silver stain or coomassie-blue stained SDS-PAGE) tubulin, e.g., Phospho-cellulose purified tubulin (Lee, J. C., Tweedy, N., S. N. Timasheff, 1978, Biochemistry, 17(14), 2783-2790), tubulin from crude cancer cell line extracts (Weatherbee, J. A., Luftig, R. B., R. R. Weihing, 1980, Biochemistry, 19 (17), 4116-4123), tubulin isolated from higher eukaryotes and their cell lines (Weatherbee et al. 1980), tubulin isolated from fungi and yeasts and their cell lines (Davis, A., Sage, C. R., Dougherty, C., K. W. Farrell, 1993, Biochemistry, 32, 8823-8835), tubulin isolated from parasitic organisms or their cell lines (Dawson, P. J., Gutteridge, W. E., K. Gull, 1983, Molecular and Biochemical Parasitology, 7(3), 267-277), and tubulins isolated from recombinant systems and recombinant organisms (Davis, A., Sage, C. R., Dougherty, C., K. W. Farrell, 1994, Science, 264, 839-842.)
Although tubulin is the target of several anti-tumor, anti-fungal and anti-anthleminitic (anti-parasitic) agents, it has not been utilized as a target in high through-put drug screening programs, diagnostic screens, or therapy because of its lability. Tubulin is unstable and will denature, becoming inactive within two days at 4xc2x0 C. (Shelanski et al., 1973.) Attempts have been made to create lyophilized forms of tubulin that are stable at room temperature. Since the first purification of tubulin by Weisenberg and Timasheff in 1970, several attempts at tubulin lyophilization have been recorded. The first by Weisenberg and Timasheff in 1970 involved crude lyophilizations on impure tubulin preparations at 97% purity (Weisenberg et al., 1970). This study did not result in assembly of competent tubulin. The second attempt in 1972 by Soifer et al. involved the reconstitution of tubulin into physiological buffer but resulted in no microtubule formation (Soifer, D., Laszlo A. H., J. M. Scotto, 1972, Biochimica et Biophysica Acta, 271, 182-192). Thus, the resulting protein was, incompetent for assembly and, therefore, inactive. Following Weisenberg et al. and Soifer et al., the dogma in tubulin biochemistry has been that highly purified tubulin cannot be lyophilized with high activity. In 1996, Sigma Chemical Company, St. Louis, Mo. (Sigma) introduced a 30-40% crude preparation of tubulin (Tubulin, Sigma Chemical Company 1996 Catalog, Catalog No. T4925). The product profile for T4925 (Lot. No. 87H4024) states that microtubules are formed upon reconstitution. However, comparative laboratory studies between T4925 and the lyophilized tubulin of the present invention disclosed herein have shown that the tubulin has very low activity as tested by a polymerization assay and very few microtubules are visible by electron microscopy. Thus, even if the Sigma formulation is sometimes successful at producing active tubulin, different batches vary widely and are inconsistent at best.
After the failures to successfully lyophilize tubulin in 1970 and 1972, Shelanski et al. in 1973 demonstrated the frozen liquid approach to tubulin storage. In this method, tubulin solution is rapidly frozen in liquid nitrogen and is stored at temperatures below xe2x88x9270xc2x0 C. Presently, this is the accepted method for storage of tubulin. This method is unsuitable for high through-put screening and other uses because retrieving the vials requires dexterity that cannot be automated easily. Another practical problem is the reproducibility of this method since the activity of preparations varies widely during the length of storage.
Prior to the invention disclosed herein, there remains a need for:
(1) lyophilized active tubulin that is highly active, stable and capable of providing reproducible results;
(2) lyophilized active tubulin that is competent of microtubule assembly;
(3) a lyophilization method for pure tubulin whereby tubulin can be lyophilized with high activity;
(4) appropriate vessels for lyophilization of tubulin so that the reconstituted form may be used for different applications; and
(5) methods of high through-put screening methods for tubulin and tubulin ligands.
The present invention provides a formulation of tubulin that is stable, i.e., it will not denature and will maintain its activity, for greater than one year at room temperature, or alternatively for greater than five years at 4xc2x0 C. This tubulin formulation is active once reconstituted and is capable of polymerizing to form microtubules. Currently available lyophilized forms of tubulin are impure (xe2x89xa640% purity), labile and unable to form microtubules. The level of stability provided by the lyophilized form of tubulin of the current invention will greatly increase the use of tubulin for applications such as research, drug discovery, diagnostics and therapy. Research applications include the possible use of the lyophilized active tubulin of the present invention to solve the three dimensional structure of tubulin (Nogales, E., Wolf, S. G., K. H. Downing, 1998, Nature, 391(6663), 199-203). The tubulin for the study conducted by Nogales et al., 1998, was supplied by Cytoskeleton, Inc. Drug discovery can be facilitated using the formulation of tubulin described in the invention as a substrate in high through-put screens for the development of new tubulin ligands. Diagnostic applications include the rapid assay of anti-tumor agents, e.g., paclitaxel, and anthlemintic agents, e.g., mebendazole. Potential therapeutic applications include the use of lyophilized tubulin in conjunction with or without anti-tubulin ligands in a complex encapsulated within a liposome. The liposome can serve as a vehicle of drug delivery.
The method of lyophilization and reconstitution described herein are breakthroughs in tubulin and tubulin ligand research and allow greater reproducibility and stability of this labile protein. Upon lyophilization, this method results in a highly active form of lyophilized tubulin that can be used with different isotypes, types, modified forms, purities, and recombinant forms of tubulin. Thus, this process is widely applicable to all forms of tubulin including microtubules formed from tubulin, MAP-rich tubulin (50% to 97% purified) (Shelanski et al. 1973), 97% to greater than 99% purified tubulin (e.g., Phospho-cellulose purified tubulin, Lee et al., 1978), tubulin in crude extracts (e.g., cancer cell line extracts), tubulin isolated from higher eukaryotes and their cell lines (Weatherbee et al. 1980; Morejohn and Fosket, 1982), tubulin isolated from fungi and yeasts and their cell lines (Davis et al. 1993), tubulin isolated from parasitic organisms or their cell lines (Dawson et al. 1983), and tubulins isolated from recombinant systems and recombinant organisms (Davis et al. 1994, Lubega et al. 1993).
The present invention covers different vessels for tubulin lyophilization since different applications of lyophilized tubulin require appropriate vessels for lyophilization and reconstitution. This includes single vials for all applications, wells in multi-well plates for high through-put screening and diagnostics, glass slides for microscopy research and diagnostics, solid supports such as dip sticks, filters, and the like, frozen drops of liquids that may be lyophilized for large quantity therapeutic applications, and any new vessels such as micro and nano-sized reaction chambers that may be available in the future.
The present invention includes the use of lyophilized active tubulin in a microtubule polymerization assay to measure the biological activity of tubulin. In this assay the optical density at O.D.340 nm is measured kinetically over time to determine polymerization and depolymerization. This kinetic assay can also be conducted at different temperatures to determine the stability of various forms of tubulin in comparison to the stable, active form of lyophilized tubulin of the present invention.
To facilitate the use of lyophilized active tubulin for the diagnostic application of assaying effective concentrations of anti-tumor agents like paclitaxel or anthlemintic agents like mebendazole, the present invention provides a high-throughput assay designed to measure anti-tubulin ligands. This assay can be used to measure ligand concentrations in body fluids such as serum, urine, and the like, fermentor broths, and to understand steps in the synthesis of ligands.
The present invention also covers potential therapeutic applications involving the use of lyophilized active tubulin, for example, the encapsulation within a microsomal liposome of a complex of tubulin and tubulin ligands to aid in drug delivery mechanisms.